We plan to test three ideas concerning the role of RNA in the control of gene expression of eukaryotic cells. The specific aims are (a) to test the hypothesis that cellular as well as viral RNA polymerase systems may use specific RNA primers to initiate transcription; (b) to define the role of specific nuclear RNA species and/or RNA subunits as cofactors in the specific cleavage of RNA precursor molecules by RNA processing enzymes; and (c) to define the role of two sorts of RNA (double-stranded RNA and small mRNA-associated RNA's) in the regulation of protein synthesis. The proposed research is so organized that the three related sets of experiments can be carried out using the same system--the HeLa cell and viruses which infect it. For RNA synthesis, we will utilize human RNA polymerase II, DNA templates such as that from adenovirus, specific HeLa cell RNA's, and our supply of HeLa cell RNA processing activities to generate potential RNA primers. For RNA processing, we will utilize HeLa subcellular fractions as sources of processing enzymes, and cellular and adeno- or influenza viral RNA transcripts, purified from cells or synthesized in vitro as substrates. For protein synthesis, the experimental system will be an mRNA-dependent protein synthesizing lysate of HeLa cells, translating viral and cellular mRNA's as assayed by ribosome protection of correct attachment sites and by the synthesis in vitro of the correct proteins.